Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Cholesterol oxidase is used commercially for the determination of serum cholesterol concentrations, is insecticidal, and is used to study membrane structure. Recent evidence suggests that cholesterol oxidase may be involved in the pathogenesis of some microorganisms such as Rhodococcus equi and Mycobacterium tuberculosis. The studies proposed will provide a precise molecular model of the lipid requirements for activity and of the pathway to flavin oxidation. These models are important for the development of cholesterol oxidase as a commercial product, as a useful and reliable tool in the study of cellular membranes for monitoring lipid rafts, for investigating the role of cholesterol oxidase in bacterial pathogenesis, and for developing anti-bacterial inhibitors. The specific hypotheses that will be tested are that 1) cholesterol oxidase can distinguish between cholesterol-containing lipid domains of different composition in the same membrane, i.e., between liquid-disordered domains and liquid-ordered domains (rafts);2) the enzyme employs a large interface to associate with the lipid bilayer during catalysis;and 3) a gated tunnel between the solvent and the flavin at the active site controls oxygen access.